Geometrically anisotropic tissues present architectural and biochemical cues critical for tissue morphogenesis and remodeling but also pose unique challenges for the field of tissue engineering. Tendons are specialized connective tissues that transmit tensile loads between muscle and bone whose functional capacity derives from native tenocytes (TCs) embedded within a highly anisotropic extracellular matrix (ECM). A primary concern for current approaches to tendon regeneration is the rapid TC de-differentiation observed in vitro, making strategies to support long-term culture of TC populations within a tissue engineered construct critically important. Further complicating this issue, like the body's response to injury, a cell's response to a biomaterial is dynamic. It is therefore important to define the initial microstructural and biochemical properties of a biomaterial but essential to control the evolution (remodeling) of these properties with time. However, the tools available to design, implement, and characterize such temporally-variable biomaterials are currently extremely limited. To address this unmet need we propose to demonstrate an innovative biomaterial approach to temporally regulate the initial presentation and remodeling of structural and biochemical cues within a model collagen biomaterial to drive long-term TC bioactivity. This approach will establish a platform for developing a functional tendon regeneration template as well as probing how the evolution of scaffold biophysical properties can be a critical design parameter for bioactive biomaterials. To accomplish these goals we have married temporally-selective biomolecule sequestration strategies with a unique fabrication process to precisely control the geometric anisotropy and structural remodeling of a clinically approved collagen-GAG (CG) scaffold system. The objective of this proposal is to demonstrate the potential for using both initial scaffold properties and subsequent remodeling as dual rheostats for optimizing TC bioactivity. Aim 1 will test the hypothesis that scaffold geometric anisotropy and strut flexura rigidity co-regulate long-term maintenance of the tenocyte phenotype. Aim 2 will test the hypothesis that temporally-organized biomolecule immobilization and release can enhance tenocyte bioactivity. Our approach represents a paradigm shift because it seeks to integrate temporal control over two key classes of extrinsic signals - matrix anisotropy as well as biomolecule sequestration and release - within a single biomaterial, making it both unique and ideally-suited for addressing our hypotheses. The capabilities and insight to be developed by this proposal do not currently exist, but offer the tantalizing potential to transform clinical treatments for a wide range of load-bearing tissue injuries through the use of temporally-ordered biomaterial substrates. Results from this project will significantly inform subsequent studies that explicitly test the regenerative potential of these biomaterials in a range of clinically-significat craniofacial and extremity tendon defects. Findings from these studies are expected to have significant impact on human health due to the large numbers of tendon repairs performed annually as well as the substantial costs, both financial and quality-of-life related, associated with failed repairs.